To approach eukaryotic gene expression and its relation to chromosome structure major emphasis will be devoted to isolating the structural gene for yeast iso-l-cytochrome c and to studying its transcription by yeast RNA polymerases I, II, and III. Cytochrome c m-RNA will be isolated by forming hybrid complexes between total yeast m-RNA and a DNA oligonucleotide complementary to the cytochrome c messenger. Yeast chromosomal DNA will be fractionated according to molecular weight and buoyant density, and those DNA fractions containing the cytochrome c structural gene will be located by their ability to hybridize to the purified m-RNA. Restriction endonuclease ecoRI will then be used to produce yeast chromosomal DNA fragments containing the cytochrome c gene which will be joined to a small E. coli plasmid, introduced into E. coli by transformation and replicated there. The isolated cytochrome c plasmid DNA will be used both to measure (by hybridization) cytochrome c gene expression and regulation and as a template for studies of in vitro transcription specificity. In a continuing study of the functional role of yeast RNA polymerases I, II, and III, yeast mutants with conditional (ts minus) defects either in RNA accumulation or DNA replication will be screened for RNA polymerase mutational alterations.